Method of producing retinyl esters

ABSTRACT

A method of producing a retinyl ester compound comprising subjecting a composition comprising retinyl or a retinyl ester and a fat or oil of animal or vegetable origins to enzyme catalysed trans-esterification in solvent free conditions to produce a retinyl ester.

This invention relates to a method of producing retinyl esters. Inparticular, it relates to a beneficial method of producing retinylesters of fatty acids from natural sources, such as fats and oils ofplant and animal origin, using enzyme catalysis. The invention may alsoprovide novel retinyl esters and oil compositions containing retinylesters, which may be useful adjuncts to cosmetic compositions.

Retinol (vitamin A) and retinyl esters have long been added to cosmeticcompositions to provide topical benefits. Retinol may typically beproduced on an industrial scale by a totally synthetic route using interalia acetone. The synthesis may generate retinyl acetate. Retinylpalmitate is another commonly used retinyl ester, which may typically beproduced by trans-esterification of retinyl acetate with methylpalmitate, with the reaction being chemically catalysed. Retinyl estershave traditionally been preferred to retinol in topical products sincethey are easier to formulate, are more stable, and they are lessirritant than the alcohol form, with the ester typically beinghydrolysed in use to the alcohol on the skin.

It is also known for retinyl esters to be produced using enzymes. Forexample, JP62248495 describes generally the production of retinyl estersfrom retinyl acetate and O-methoxypolyethylene glycol modified lipasesto produce long chain acid esters. This application also describes theproduction of retinyl oleate by similarly modified enzymes in a mediumcomprising benzene saturated with water. However, in this teaching, thereactions between the vitamin A and the long chain fatty acids occur inorganic solvent systems, such as e.g. benzene. Also, as the modifiedlipase is solubilised in the reaction system, significant furtherprocessing is required to separate the desired retinyl ester productfrom the reaction mixture.

It is also known from the Australian Journal of Chemistry, 45 (4)641-649(1992), O'Connor C. J; Petricevic S. F and Stanley R. A., that C.rugosa can be used in the production of retinyl palmitate from freealcohol and fatty acid in aqueous ethanol or biphasic mixtures ofparaffin and water. Again, this process requires the use of organicsolvents.

In addition, WO 99/32105 (DCV, Inc) describes the production ofconjugated linoleic acid esters in the presence of a lipase, whichesters can include retinyl esters. However the application does notstate how the retinyl esters were obtained and the examples shown forother esters require the use of solvent and significant processing toisolate the final products.

It is also suggested in Journal of Molecular Catalysis B: Enzymatic, 8,275-280 (2000) (Maugard T; Legoy M. D) that retinyl esters may beproduced by enzymatic routes. The paper describes the production ofretinyl adipate, succinate, oleate and lactate for incorporation intocosmetic products. Again the retinyl esters are prepared in solventsusing enzymes such as Candida antarctica or Rhizomucor miehei. However,again this document suggests that a solvent is necessary for thereaction to be carried out, and does not discuss the nature or source ofthe acyl donor.

The present invention aims to provide a new method of preparing retinylesters for use e.g. in topical cosmetic compositions, which esters mayhave various benefits associated with them over prior art teachings,including being simpler and cheaper to produce, without the requirementfor organic solvents or significant down-stream processing. Surprisinglythe products of the invention also show much enhanced stability, andreduced irritancy on the skin.

Thus, according to a first aspect of the invention, there is provided amethod of producing a retinyl ester compound comprising subjecting acomposition comprising retinol or a retinyl ester and a fat or oil ofanimal, vegetable or algal origin to enzyme catalysedtrans-esterification in solvent free conditions to produce a retinylester.

In a further aspect, there is provided a composition comprising a fat oroil of animal, vegetable or algal origin containing retinyl esters offatty acids contained in the animal, vegetable or algal fat or oil. Theretinyl esters are preferably formed by enzyme catalysedtrans-esterification.

In yet a further aspect, there is provided a retinyl ester of a fattyacid prepared by the method described above.

According to yet a further aspect there is provided a topicalcomposition for application to human skin containing a retinyl ester ora composition containing a retinyl ester prepared as described above.

According to yet a further aspect there is provided a cosmetic method oftreating human skin comprising applying thereto a topical composition asdescribed above.

According to yet a further aspect of the invention, there are providednovel retinyl fatty acid esters, such as (but not necessarily limitedto) retinyl C18:3 and C18:4 conjugated fatty acid esters.

The method may be used to provide compositions containing a fat or oilof animal, vegetable or algal origin, and which contain (or from whichmay be isolated) retinyl esters with fatty acid portions which reflectthe fatty acid composition of that animal, vegetable or algal fat oroil. For example, when produced in sunflower oil, the method producessunflower fatty acid retinyl esters from the enzyme catalysedtrans-esterification of sunflower oil. The resultant retinyl esters arepredominantly the linoleic and oleic forms, reflecting the fatty acidcomposition of the sunflower oil.

The method can be extended to the use of any fat or oil of animal,vegetable or algal origin.

As a result, the method can be used to synthesise retinyl esterscontaining fatty acids having C₁₂₋₂₂ chain lengths, either saturated orunsaturated. The resulting retinyl esters and retinyl ester blends havebeen found to be relatively mild compared to e.g. retinyl acetate orretinyl palmitate made by conventional routes. The method also providesa route to the manufacture of retinyl esters and ester blends from arelatively cheap starting material, retinyl acetate, which is cheaper toprepare than materials such as retinyl palmitate.

As an enzyme to be used for the trans-esterfication process, preferablya lipase enzyme is used. Lipase enzymes are well known for their abilityto catalyse (trans) esterification reactions involving oils and fats.Any suitable source of lipase can be utilised, though industriallyproduced lipases are preferred on a cost basis. The lipase shouldpreferably be immobilised on a suitable carrier.

The fat or oil of animal, vegetable or algal origin is in fact acomposition which contains either a free fatty acid, or an ester offatty acids which are of animal, vegetable or algal origin. Any such oilor fat, being of natural origin will typically contain a population offree fatty acids acids or esterified fatty acids, although one or moremay predominate in this population. However the population of free fattyacids or esterified fatty acids will typically characterise the animal,vegetable or algal source.

Preferably, a fat or oil of animal, vegetable or algal origin is chosensuch that the fatty acid content of the oil or fat of the hydrolysedesters in the oil or fat is relatively enriched in skin benefit agents.Preferred skin benefit agents for the oil or fat may be relatively highin (i.e. contain more than about 0.1%, preferably 0.5%, more preferablymore than about 1.0% of) include C18:1-C22:6 fatty acids, particularlypetroselinic acid or conjugated 18:2, 18:3 and 18:4 acids.

The invention has a number of benefits and distinguishing features overthe prior art.

Firstly, in the process of the invention, retinol is trans-esterifiedwith free fatty acids or more likely (and preferably) fatty acid estersin the oil or fat of animal, vegetable or algal origin. As a result,fatty acid side chains which were previously present in the reactionmixture as a fatty acid side chain in an ester such as a fatty acidtriglyceride are now present as part of a retinol ester. Since theretinol ester is hydrolysed on the skin, retinol and the fatty acid arereleased.

If the fatty acid is also a known skin benefit agent such as e.g.outlined above, on hydrolysis the skin is treated to a “double dose” ofskin benefit agents, namely the retinol and the fatty acid, as well aspossibly receiving some extra skin benefit from the fat or oil ofanimal, vegetable or algal origin itself. Fatty acids found in oils orfats of animal, vegetable or algal origin, especially in thetriglycerides found therein, may provide a relatively cheap source ofsuch benefit agents. As such, an oil or fat used according to theinvention may be selected for its fatty acid profile (either in the formof free fatty acids or triglycerides).

In addition, since the trans-esterification reaction is carried outdirectly in the fat or oil of animal or vegetable origin in solvent freeconditions, which fat or oil may itself have skin beneficial properties,there is no need to conduct any subsequent clean up operation either toremove solvents from the reaction mixture or to isolate or concentratethe retinyl ester. Instead, the fat or oil containing the retinyl estermay be either applied directly to the skin, or dosed into a topicalcomposition for application to the skin. To this end though, it ishighly preferred that the enzyme used in the trans-esterification beimmobilised on a solid support in such a way that it can be readilyremoved from the reaction mixture after trans-esterification, forexample by filtration.

Such immobilisation techniques are well known in the art, and includefor example immobilisation on microporous polypropylene beads which havebeen pre-treated with surfactant, to which is added an enzyme solutionand which is subsequently washed and dried.

The enzyme may be used in normal functional conditions for that enzyme,which typically include simple mixing of the enzyme in a container withthe other reactants, at temperatures of up to about 70° C.

As mentioned above the trans-esterification reaction is carried out insolvent free conditions. By this is meant that the composition containsless than about 10% solvent, preferably less than about 5% solvent, morepreferably less than about 1% solvent, and preferably is totally solventfree.

Solvents which are excluded from the transesterification reactioninclude water (the only water present is preferably only that which isassociated with the enzyme itself), as well as short chain (i.e C₁₋₆)solvents such as alkanes and alcohols, ketones and any esters which mayinterfere with the desired trans-esterification reaction.

The tranesterification reaction should take place in a medium which hasa significant liquid phase, e.g. is a liquid or a paste.

Many preferred fatty acids which are the subject of thetrans-esterification reaction are liquid at room temperature; many alsocontain a degree of unsaturation.

Retinyl esters produced according to the invention which have a fattyacid profile based on the fatty acid content of the correspondinganimal, vegetable or algal oil or fat have been found to havesurprisingly good physical stability compared to other single speciesfatty acid esters, as well as be surprisingly mild.

The resultant transesterified esters reflect the fatty acid chain lengthcomposition of the animal or vegetable oil used in the method. Henceexemplary retinyl esters which may be prepared according to theinvention are prepared using the following plant and animal oils, andmay have enriched fatty acid ester contents as outlined below:

-   C12:0—coconut oil, palm kernal-   C14:0 and C14:1—kombo nut (Pycnanthus angolensis) oil-   C16:0—palm oil-   C18:0 and C18:1—cocoa butter-   C18:1—high oleic sunflower oil, olive oil, coriander seed oil-   C18:1 and C18:2—corn oil, sunflower oil, cotton seed oil-   C18:2—safflower oil, grape seed oil, wheatgerm oil-   C18:3—borage oil, evening primrose oil, linseed oil, pine nut oils,    Manketti nut oiland pomegranate seed oil-   C18:4—Impatiens balsamina seed oil-   C20:5 and C22:6—fish oils, algal oils-   C22:1—crambe oil, mustard seed oil

Preferred retinyl esters include those prepared from the fatty acids ofplant oils, especially kombo nut oil, coriander oil, sunflower oil,safflower oil, pomegranate oil, borage oil and pine nut oil, as well asretinyl esters of conjugated fatty acids, especially C18 conjugatedfatty acids. Unless otherwise purified the retinol esters which are thesubject of the invention will invariably comprise a range of fatty chainlengths and types which reflect the fatty acid content of the fat or oilof animal or vegetable origin, or more likely its triglyceride fattyacid content.

Preferred species of retinyl conjugated fatty acid esters which arebelieved to be novel in their own right include:

-   -   retinyl ester of punicic acid (18:3, c9, t11, c13), which can be        prepared from pomegranate seed oil    -   retinyl ester of calendic acid (18:3, t8, t10, c12), which can        be prepared from Calendula officinalis (Marigold) seed oil.    -   retinyl ester of eleostearic acid (18:3, c9, t11, t13), which        can be prepared from Manketti nuts (Ricinodendron rautanenii or        Ricinodendron heudelotti), but which can also be prepared from        cherry kernal oils (Prunus Cerasus, P. avium, P. mahaleb), tung        oil, Momordica dioica (Chinese bitter melon), Parinari montana        and Parinarium excelsis.    -   retinyl ester of parinaric acid (18:4, c9, t11, t13, c15), which        is preferably sourced from Impatiens balsamina L, but may also        be sourced from Parinari glaberrimum, Lithospermum euchromum,        Sebastiana brasiliensis, I. Edgeworthii, I. Pallida & capensis        and Parinarium laurinum.

The enzyme used in the method according to the invention is preferably alipase enzyme, more preferably a lipase immobilised on a solid support.Examples of suitable lipase enzymes include Candida rugosa lipase,Lipase D and Lipozyme IM. Methods of immobilisation of lipases (whererequired) are described, for example, in EP424130.

Topical compositions for application to the human skin preferablycomprise 0.00001 to 5%, preferably 0.0001 to 1%, more preferably 0.01 to0.5% of the retinyl esters prepared according to the invention.

The invention will now be prepared by way of example only with referenceto the accompanying drawings, in which:

FIG. 1 shows the stability of retinyl esters according to the inventioncompared to commercially available retinyl palmitate and retinyllinoleate.

FIG. 2 shows the comparative stability of esters according to theinvention in topical products, and

FIG. 3 shows the relative irritancy of retinyl ester formulations.

EXAMPLE 1 Production of Retinyl Esters Using Sunflower Oil

Sunflower oil (7.5 g) and retinyl palmitate (2.5 g) were mixed and water(0.03 g) and immobilised Lipase D (Rhizopus oryzae (Amano) immobilisedon Accurel® EP100 macroporous polypropylene (Acordis) 0.1 g) or Candidarugosa AY (Amano) on Accurel® EP100 (Acordis) (CR; 0.1 g) added. Themixtures were placed in a shaking water bath at 55° C. for 20 or 23hours. The immobilised lipase was then removed by simple filtration todirectly yield the product, a solution of retinol esters in triglycerideoil. The composition of the retinyl esters was separated from thetriglycerides by thin layer chromatography (0.5 mm silica G plates,Analtech Ltd.) using toluene as eluting solvent and visualised byspraying with 1% 2,7-dichlorofluorescein in ethanol. The ester band wasscraped off and FAMEs were produced using 3 ML sodium methoxide and 1 mLtoluene at 80° C. for 20 minutes followed by 5 minutes at 80° C. withboron trifluoride (2 mL volumes of reagent). The fatty acid content wasthen analysed by Fatty acid methyl ester Gas Chromatography (FAME-GC).GC conditions: Column—30 m/0.53 mm/0.5 um restek famewax, Helium carriergas 15 kpa, flame ionisation detection, 260° C. PTV injection (Run: 3min @ 260° C., 80° C. hold 1 min, +20/min to 180° C., +2/min to 220° C.,+1/min to 230° C., +4 min hold at 230° C.).

As shown in Table 1, the palmitic acid content of the retinyl ester wasreduced from 98.5%, being replaced predominantly by linoleic and oleicacids. The palmitic acid was incorporated into the sunflowertriglycerides

TABLE 1 Sunflower retinyl esters produced from retinyl palmitate C18:2C18:1 (lino- C18 Sample C14:0 C16:0 C18:0 Other (oleate) leate) (tot)Retinyl 0.4 98.5 0.7 0.4 0 0 0.7 palmitate starting material Retinylester 0.2 44 5.1 0.4 16.9 33.4 55.4 product (CR - 23 h) Retinyl ester0.2 28.9 5.1 1.7 21.2 42.9 69.2 Product (LD - 20 h)

EXAMPLE 2 Production of Sunflower Oil Retinyl Esters from RetinylAcetate

Sunflower oil (7.5 g) and retinyl acetate (3.75 or 2.5 g) were mixed andwater (0.03 g) and 1% immobilised Rhizomucor miehei lipase (Lipozyme IMNovo Nordisk) added. The mixture was placed in a shaking water bath at55° C. for 4 days. The immobilised lipase was then removed by simplefiltration to directly yield the product, a solution of retinol estersin triglyceride oil. The levels of retinyl esters were determined bypeak collection from HPLC, with the addition of C17 methyl ester ISTDaccording to the following conditions:

-   Column: 10 cm Nucleosil 100A 3 um silica with precolumn.

Elution solvents—Solvent A—hexane/toluene 1:1; Solvent B—toluene/ethylacetate/formic acid 600/200/16; solvent C—toluene/ethylacetate/isopropyl alcohol/formic acid 500/200/100/16.

Time (Mins) Solvent ratios (A/B/C) 0 99/1/0 4.9 99/1/0 5 90/10/0 675/25/0 7 40/60/0 9 10/90/0 9.1 0/10/90 12 0/10/90 12.1 10/90/0 15.010/90/0 15.1 99/1/0 30.0 99/1/0

-   Flow rate: 1.4 mL/min-   Detection: Evaporative light scattering detector (40° C./10 L/min    nitrogen)

The retinyl ester levels produced are shown in Table 2. The retinylesters collected from the HPLC were converted to FAMEs using 2 ML sodiummethoxide at 80° C. for 20 minutes. FAME-GC was performed as describedpreviously (Table 3).

TABLE 2 Retinyl ester levels % Long chain Description retinyl esters 2:1SF:RA Lipase SP392(supported) 4 20.4 days 3:1 SF:RA LipaseSP392(supported) 4 17.3 days

TABLE 3 Fatty acid profile of retinyl esters Fatty acid chain length 2:1SF:RA 3:1 SF:RA 16:0 14.2 12.7 16:1 0.9 3.4 18:0 5.1 5.0 18:1 22.6 22.218:2 55.1 54.3 18:3 0 0.3 20:0 0.8 0.7 20:1 0.8 0.1 22:0 0.5 0.9 22:1 00.4

EXAMPLE 3 Retinyl Linoleate Stability in Moisturising Cream Formulations

Retinyl Linoleate from National Starch Corp. and the retinyl esterproduct from example 1 were blended into moisturising cream formulationsshown below in Formulations 1 and 2 at a level of 0.03 and 0.15% of theretinyl ester. The creams were stored at 22° C., 30° C., 45° C. and 50°C. and samples taken at time 0, and twice more over 35 days as shown inFIGS. 1 and 2.

Formulation 1

Ingredient Name % Supplier Phase A Water to 100 Local Water, Spring 1.00Local, Poland Springs Glycerin 5.00 Dow Disodium EDTA 0.05 W R. GracePanthenol 0.10 Roche Mg Amino Acid Chelate 0.01 Maypro Zn Amino acidChelate 0.01 Maypro Green Tea extract 0.10 Tri-K Grapeseed Extract 0.10Brooks Phase B Ultrez 10 0.75 B F Goodrich TEA 99% 2.00 Dow Parsol HS2.00 Roche Water 10.00 Local Lanett 14 (Myristal 0.50 Cognis Alcohol)Arlacel 60 (Sorbitan 1.20 ICI Stearate) Cetyl Alcohol 0.50 HankelEmulsynt GDL (Glycerol 0.50 ISP Dilaurate) Stearyl Alcohol 0.50 RTDRyoto Sugar Ester 0.25 Ryoto Myrj 59 (PEG-100 Stearate) 0.50 ICIPristerene 4911 (Stearic 0.25 Stephan Acid) Parsol MCX (Octyl methoxy4.322 Roche cinnimate) Parsol 1789 (Butyl Methoxy 2.00 Roche dibenzoyl .. . ) Dermablock OS (Octyl 3.90 Alzo Salicylate) Vitamin E Acetate 0.10Roche Retinyl linoleate-National 1.5 Starch Incromide LSM Linoleate 0.01Croda Cholesterol NF 0.10 Croda BHT 0.02 Tocomix 0.003 Permethyl 101A4.50 Presperse Phenonip 0.54 Clariant Phase C Vitamin A Palmitate 0.01Roche Fragrance Q26913 0.20 Quest TOTAL 100.00

Formulation 2

Ingredient Name % Supplier Water to 100 Local Water, Spring 1.00 Local,Poland Springs Glycerin 5.00 Dow Disodium EDTA 0.05 W R Grace Panthenol0.10 Roche Mg Amino acid Chelate 0.010 Maypro Zn Amino acid chelate0.010 Maypro Green Tea Extract 0.100 Tri-K Grape seed extract 0.100Brooks Phase B Ultrez 10 0.75 B F Goodrich TEA 99% 2.00 Dow Parsol HS2.00 Roche Water 10.00 Local Lanett 14 (Myristal Alcohol) 0.50 CognisArlacel 60 (Sorbitan Stearate) 1.20 ICI Cetyl Alcohol 0.50 HankelEmulsynt GDL (Glycerol Dilaurate) 0.50 ISP Stearyl Alcohol 0.50 RTDRyoto Sugar Ester 0.25 Ryoto Myrj 59 (PEG-100 Stearate) 0.50 ICIPristerene 4911 (Stearic Acid) 0.25 Stephan Parsol MCX (Octyl methoxyCinnimate . . . ) 4.577 Roche Parsol 1789 (Butyl Methoxy dibenzoyl . . .) 2.00 Roche Dermablock OS (Octyl Salicylate) 3.12 Alzo Retinyllinoleate (from example 1) 1.5 Incromide LSM (Linoleamide MEA) 0.01Croda Cholesterol NF 0.10 Croda BHT 0.02 Tocomix 0.003 Permethyl 101A4.50 Presperse Phenonip 0.54 Clariant Phase C Fragrance Q26913 0.20Quest Vitamin A Palmitate 0.01 Roche TOTAL 100.00

The retinyl esters were extracted from the base cream 60/40acetone/acetonitrile, stirred for 10 minutes, filtered and then injectedThese were then analysed by HPLC according to the conditions below.

-   Column: Intersil 5 micron, ODS-2 (C18) from Phenominex-   Detection: UV at 325 nm-   Mobile phase: 35% Acetone/65% Acetonitrile-   Flow Rate: 1.0 mL/minute-   Peak Elution: Approx 10 minutes-   Calib Curve: Standards—1.5 ppm to 18 ppm for samples containing 0.03    to 0.15% RL.

FIGS. 1 and 2 show the stability results for the samples. The retinylester product from example 1 was more stable than the National Starchoriginating retinyl linoleate. For comparison retinyl palmitate is onlyas stable as retinyl linoleate (National Starch).

EXAMPLE 4 Stability of Retinyl Esters in Oils

The stability of retinyl linoleate (National Starch) and retinyl esters(example 1) in the fluid oil phase alone of a moisturising creamformulation as evaluated. Two fluid oil formulations were prepared byincorporating the desired amounts of the ingredients like Parsol MCX,Dermablock OS, Permethyl 101A and Vitamin E acetate as found inFormulation 1. One was spiked with retinyl esters (example 1) and theother with retinyl linoleate from National Starch (NS) at the 0.75%level. These were analysed initially and after twenty eight days at 50°C. (Table 4)

TABLE 4 Stability of retinyl esters in the oil phase Retinyl esters %recovery Retinyl linoleate 72.6 (NS) Retinyl esters 85.0 (Example 1)

The data demonstrates that retinyl esters from example 1 are more stablein oil blends than retinyl linoleate.

EXAMPLE 5 Irritancy of Retinyl Esters

5 male and 15 female subjects were selected for the study, the aim ofwhich was to compare the irritation potential of a moisturising creamalone and in combination with retinyl linoleate from example 1 visNational Starch material. The concentration of retinyl linoleate addedto the moisturising cream from the 2 sources contained equal molarquantities of the retinyl moiety in ester form. Test materials werecompared to a selected control statistically in a pairwise comparisonbased on Friedman's Rank Sums (p<0.10 is considered significant, pvalues 0.2 and 0.15 are provided for informational purposes only).

Protocol

The upper outer aspect of the upper arm was exposed to the testmaterials once for a 24 hour period followed by up to three 18 hourexposures, using 25 mm Hill Top® Chambers fitted with 18 mm Webril®padding and held in place with Scanpor® tape. 0.2 ml of the testmaterials was applied to the Webril® padding 1 hour before application.

Dose Characterization/Verification and Stability and Microbiology:

The level of Retinyl linoleate was within target for all Test materialscontaining this material. All Test materials were found to bemicrobiology acceptable.

As shown in FIG. 3, the retinyl linoleate from example 1 (RL EX1) is farless irritant than the retinyl linoleate from National starch (RL NS).

EXAMPLE 6 Production of Retinyl Esters from Naturals Oils and RetinylAcetate

Retinyl esters were prepared using natural oils including Sunflower oil,coriander oil and hexane extracts of pomegranate, Impatiens balsimina,and Manketti seeds. The oils and retinyl acetate were mixed in a ratio3:1 (7.5 g:2.5 g) with water (0.03 g) and either 1% immobilisedRhizomucor miehei lipase (Lipozyme IM Novo Nordisk) or Lipase D. Themixture was placed in a shaking water bath or stirred vessel at 55° C.for 4 days. The immobilised lipase was then removed by simple filtrationto directly yield the product, a solution of retinol esters intriglyceride oil. The retinyl esters were analysed using solid phaseextraction (SPE) column separation followed by gas chromatography (GC)as described below.

Analysis method: 20 mgs of retinyl ester/oil along with a methyl esterinternal standard were dissolved in 0.5 mL hexane and loaded onto a 0.5g silica SPE column. Retinyl esters and the methyl ester internalstandard were eluted with 10 mL 2% diethyl ether in hexane. Retinylesters were converted to methyl esters and the fatty acid compositionquantified against by GC.

TABLE 4 Analysis of retinyl esters Fatty acid chain CorianderPomegranate Manketti Impatiens length esters esters esters esters 16:026.3 27.0 42.9 35.5 18:0 1.8 2.6 5.4 4.9 18:1 59.6 4.4 4.3 9.6Petroselinic 18:2 8.4 3.9 8.9 6.3 18:3 52.3 32.4 11.3 PunicicEleostearic 18:4 16.3 Parinaric C18 total 69.8 68.5 51.3 51.5 other 3.99.8 6.1 16.1

1. A method of producing a retinyl ester compound comprising subjectinga composition comprising retinol or a retinyl ester and a fat or oil ofanimal or vegetable origins to enzyme catalysed transesterification insolvent free conditions to produce a retinyl ester.
 2. A methodaccording to claim 1 wherein the fat or oil of animal or vegetableorigin contains one or more C₁₂₋₂₂ saturated or unsaturated fatty acids.3. A method according to claim 1 wherein the enzyme is a lipase enzyme.4. A method according to claim 1 wherein the enzyme is immobilised on asupport.
 5. A method according to claim 1 wherein the fat or oil ofanimal or vegetable origin contains a fatty acid triglyceride.
 6. Amethod according to claim 1 wherein the source of the fatty acid iskombo nut oil, coriander oil, sunflower oil, safflower oil, pomegranateseed oil, Manketti nut oil, fish oil, borage oil, pine nut oil orImpatiens balsamina seed oil or calendula seed oil.
 7. A methodaccording to claim 1 wherein the enzyme is immobilised on a solidsupport.